qPCR and Digital PCR Conference in London
The qPCR and Digital PCR Conference started with lectures on the reliability ofqPCR and ddPCR, from their technical limitations to the interpretation of results to errors in published data with a very interesting presentation by Prof. Stephen Bustin (data will be published in 2017 – details prohibited at the moment).
Details of the different presentations will be available short term after the speakers gave their permission for publication. As soon as the presentations are available we will place them on the Genaxxon bioscience internet page or you can ask us at: firstname.lastname@example.org.
The talks started with Prof. Stephen Bustin from Anglia Ruskin University. The quintessence of his lecture was that approximately 90% of screened peer publications (data presented in London) about qPCR > contain technical or procedural errors, such as wrong primer and DNA sequences, incorrectly given cell lines, or even results, for example, whether a gene is high - or down-regulated. A repetition of the published data was, in some cases, only possible after corresponding procedures are adapted, changed or completely changed by Prof. Bustin's team. Prof. Bustin and his team worked out guidelines (published in Nature Methods) for minimum standards encompassing the key assay parameters for accurate design, documentation and reporting of qPCR experiments (MIQE précis >), which he will present on a webinar >.
As a further point of failure he named the use of so-called hotstart polymerases > or hotstart master mixes >. An investigation of more than 10 different master mixes from well-known manufacturers showed that the respective polymerase employed showed a clear activity if a completely pipetted master mix was simply allowed to stand for a longer time at +4°C.
Comment from Prof. Bustin: “Most investigated polymerases show significant non hotstart activity and some seem to be more or less pure standard Taq polymerases >!”
For this reason data produced with such hotstart polymerases (master mixes) are not reliable and reproducible as amplification depends strongly on the operator and the set-up.
The following lecture by Jim Huggett (Principal Scientist at LGC, UK) dealt with the reliability and reproducibility of ddPCR compared to qPCR. To investigate this, different samples were given to 21 different laboratories. These laboratories did not receive any detailed information about the samples and should simply examine the delivered samples according to local operation procedures to determine the original amount of DNA. In contrast to corresponding tests carried out on the basis of qPCR, the result was convincing. 18 out of 21 laboratories came to identical results. The equipment and chemicals used were not calibrated beforehand!
Dr. Jim Huggett was able to demonstrate that ddPCR is a very reliable method for the detection and quantification of DNA, which makes it possible to make absolutely and unambiguously clear statements about the content of specific target DNA in a mixture of different DNA targets and to verify these results between different laboratories which is not possible if qPCR > is used as the examination/verification technique.
The advantages of ddPCR are particularly evident in the detection of "minority populations" as in HIV-1 or the detection of malaria in persons returning from a malaria region to malaria-free regions, in the detection of low copy number genes and SNPs.
Despite all, there are, of course, still disadvantages of ddPCR. Above all these includes the high costs, the low throughput and the physical sensitivity or the dynamic range.
Prof. Lieven Clement from the University of Ghent talked about mathematical considerations regarding the evaluation of data produced by ddPCR.
In case of ddPCR evaluation of raw data can only be done using the Poisson equation (https://en.wikipedia.org/wiki/Poisson_distribution). The Poisson equation enables, based on the raw data, to calculate how much starting material (DNA) was actually present in the sample. https://www.youtube.com/watch?v=9TJy_uegmd4
For getting reliable results Lieven stressed on the fact that one of the most important factors is the determination of the right threshold which depends pretty much on the experiment, respective the amount of target DNA in the sample material.
Maurice van den Hoff, Associate Professor University of Amsterdam showed data about his work on the Wnt signaling pathway about wrong results that are produced in case of low copy numbers due to artefacts in case of using an unspecific fluorescent dye >. Using an unspecific fluorescent dye will most likely result in wrong (artificial) data if Ct-values are above 28 to 30 and if the used primers are not really optimal designed.
The results of Maurice showed excellent qPCR curves in any case but additional evaluation by Agarose gel electrophoresis and temperature gradient qPCR revealed that more than 50% of all data with Ct-values above 28 seem to be excellent but are questionable if not evaluated very carefully by additional detection methods.
Bustin, S.A., Beaulieu, J.F., Huggett, J., Jaggi, R., Kibenge, F.S., Olsvik, P.A., Penning, L.C., Toegel, S. (2010) MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol. 11:74
Stephen A Bustin, Vladimir Benes et al.: The need for transparency and good practices in the qPCR literature. Nature Methods 10, 1063–1067 (2013) doi:10.1038/nmeth.2697, Last updated: 9 November 2016 7:58:44 EST
Devonshire AS, O'Sullivan DM, Honeyborne I, Jones G, Karczmarczyk M, Pavšič J, Gutteridge A, Milavec M, Mendoza P, Schimmel H, Van Heuverswyn F, Gorton R, Cirillo DM, Borroni E, Harris K, Barnard M, Heydenrych A, Ndusilo N, Wallis CL, Pillay K, Barry T, Reddington K, Richter E, Mozioğlu E, Akyürek S, Yalçınkaya B, Akgoz M, Žel J, Foy CA, McHugh TD, Huggett JF. The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis. BMC Infect Dis. 2016 Aug 3;16:366. doi: 10.1186/s12879-016-1696-7.